generation-assembly-virus-specific-nucleocapsids-respiratory

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and ...

Emory University School of Medicine

The templates for transcription and replication by respiratory syncytial virus (RSV) polymerase are helical nucleocapsids (NCs), formed by viral RNAs that are encapsidated by the nucleoprotein (N). Proper NC assembly is vital for RSV polymerase to engage the RNA template for RNA synthesis. Previous studies of NCs or nucleocapsid-like particles (NCLPs) from RSV and other nonsegmented negative-sense RNA viruses have provided insights into the overall NC architecture. However, in these studies, the RNAs were either random cellular RNAs or average viral genomic RNAs. An in-depth mechanistic understanding of NCs has been hampered by lack of an  assay that can track NC or NCLP assembly. Here we established a protocol to obtain RNA-free N protein (N) and successfully demonstrated the utility of a new assay for tracking assembly of N with RNA oligonucleotides into NCLPs. We discovered that the efficiency of the NCLP (N-RNA) assembly depends on the length and sequence of the RNA incorporated into NCLPs. This work provides a framework to generate purified N and incorporate it with RNA into NCLPs in a controllable manner. We anticipate that our assay for  trackable assembly of RSV-specific nucleocapsids may enable in-depth mechanistic analyses of this process.

 trackable assembly of RNA-specific nucleocapsids of the respiratory syncytial virus.

The M2-1 protein of human respiratory syncytial virus (HRSV) is a transcription anti-terminator that regulates the processivity of the HRSV RNA-dependent RNA polymerase (RdRP). Here, we report a crystal structure of HRSV M2-1 bound to a short positive-sense gene-end RNA (SH7) at 2.7 Å resolution. We identified multiple critical residues of M2-1 involved in RNA interaction and examined their roles using mutagenesis and MicroScale Thermophoresis (MST) assay. We found that hydrophobic residue Phe23 is indispensable for M2-1 to recognize the base of RNA. We also captured spontaneous binding of RNA (SH7) to M2-1 in all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method. Both experiments and simulations revealed that the interactions of RNA with two separate domains of M2-1, the zinc-binding domain (ZBD) and the core domain (CD), are independent of each other. Collectively, our results provided a structural basis for RNA recognition by HRSV M2-1.

Claire Ogilvie

Department of Biochemistry

Emory University School of Medicine

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus

Claire Ogilvie

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biochemistry

Emory University School of Medicine

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus

Department of Biochemistry